Incorporating Quantitative Polymerase Chain Reaction (qPCR)

Analyses into Site Management

Kirsti M. Ritalahtikrita@utk.edu

Introduction

Value of quantifying bacteria, their function and activity

Molecular Biological Tools (MBTs) and Environmental Molecular Diagnostics (EMDs)

Quantitative Real-Time PCR

Available Assays

Application to MNA

Value of Quantitative Microbial Information via Molecular Tools

Dilute and culture on agar

Count colonies

Stain and Count by Epifluorescence

Microscopy

Count cells

Enumerating bacteria by traditional means is tedious

Molecular tools expedite analysis, provide quantitative information, and can target bacteria that cannot be grown in isolation

We cannot study bacteria in isolation andexpect to see their behavior in nature

What are MBTs and EMDs

Molecular Biological Tools (MBTs)

Tools targeting biological function in an environment, that can be used to enumerate, quantify or inform on the potential to carry out some desired function

Environmental Molecular Diagnostics (EMD)

Utilizing the information from the MBTs to effectively predict, and manage contaminated sites or other microbial processes

Benefits of MBTsSpecific, sensitive, quantitative information regarding organisms

and genes involved with bioremediation

Provide information about the relevant processes affecting contaminant longevity

Applicable to a wide variety of environmental samples

Predictive tool for site assessment

Is MNA (monitored natural attenuation) an option?

Is biostimulation sufficient?

Is bioaugmentation necessary?

What endpoints can be expected?

Targets in a Cell

mRNA DNA

Lipid

Activity

rRNA

Ribosome and rRNA

Protein

MBTs

Protein

RNA

DNA Potential, gene numbers

General metabolic activity

Specific metabolic activity

Direct measure of activity

Lipids General metabolic activity

rRNAmRNA

qPCR, TRFLP, DGGE, FISH

RT-qPCR, CARD-FISH

CSIA, proteins,enzyme probes

PLFA

Quantitative Real-Time PCR (qPCR)

qPCR is a robust assay for quantifying nucleic acid targets in environmental samples

Both DNA and RNA can be analyzed in most samples

Used for site assessment and long term monitoring programs

Dynamics in gene abundances and gene activity (i.e., transcripts) serve as activity indicators

Correct QC protocols must be established from sample collection through qPCR analysis

Ideal QC adjusts for losses in target number during sample collection, shipping, preparation and analysis

F MGBQ

F MGBQ

qPCR

1. Hybridization

F Primer

R Primer

TaqMan Probe

Template

2. Strand Displacement, Taq Polymerase Binds

F Primer

R Primer

Template

Taq

F MGBQ

qPCR3. Primer Extension

4. Probe Displacement and Flurophore Release

F Primer

R Primer

MGBQ

Template

Taq

F

F Primer

R Primer

Template

Taq

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cycle

Nor

mal

ized

fluo

resc

ence

0.1

1.0

10.

0

qPCR

5. Fluorescence Curves for Standards

Ct

qPCR

CT 17.5Log Dhc= 7.116S rRNA copies= 1.3 x 107

CT 34.5Log Dhc= 1.816S rRNA copies= 6 x 101

6. Analysis based on standard curve

Many Available Assays

Target microbial activities relevant to bioremediation and other environmental processes:

Total Bacteria, Archaea, Methanogens

Reductive Dechlorination/Aerobic Oxidation

Petrolium Detoxification

Nitrogen Cycling

Microbially Induced Corrosion

Metal/Radionuclide Reduction

Commercial Assays

qEBAC “General Bacteria”

qARC “General Archaea”

qAPS Sulfate Reducing Bacteria

qMGN Methanogens

qDHC DehalococcoidesqTCE TCE to cDCEqVC cDCE and VC to ethene

qDHB Dehalobacter

qDHG Dehalogenimonas*

qDSB Desulfitobacterium

Target microbial activities relevant to bioremediation and other environmental processes:

qRMO ring hydroxylating toluene monnox.

qPHE phenol hydroxylase: BTEX oxidation

qTOL tol monooxygenase: toluene, xylene

qBSS benzylsuccinate synthase: toluenexylene

qTOD tol dioxy: benzene toluene ethylbenzene

qNAH prnapthalene

qPM1 MTBE utilizing Methylibium PM1

qDSM Desulfuromonas

qGEO Geobacter

qAGN acid producing bacteria (MIC)

qARG archaeglogus (MIC)

qDNF nitrate reducing bacteria

qpcrA perchlorate reductase

qAOB ammonia oxidizers

qsMMO methane monooxygenase

qPPO propane monooxygenase

qBOM butane monooxygease

PCE TCE cis-DCE

Example: Chloroethene Dechlorination

Dehalobacter, Desulfuromonas, Sulfurospirillum,

Geobacter (et al.)

pceA

For the rest of the story go to the MI website and watch the seminar from

Frank Loeffler on Feb. 11, 2013

Dehalococcoides: Keystone Bacteria for Detoxification

PCE TCE cis-DCE VC Ethene

tceA bvc

A

pceA

vcrA

Five Dhc Gene Targets (Biomarkers)1. Dhc 16S rRNA gene2. pceA encoding a PCE to TCE RDase3. tceA encoding a TCE-to-VC RDase4. bvcA encoding a VC-to-ethene RDase5. vcrA encoding a VC-to-ethene RDase

Dhb 2

Dhb 1

Dhb 3

dcrA

cfrA

cfrA

pceA

pceAtceA

tceAbvcAvcrA

bvcAvcrA

Dhc

Dhc

Dhb 1

GeoDsmDhbDhc

Anaerobic Dechlorination Biomarkers

???

Chlorinated Ethanes Chlorinated Ethenes Chlorinated Methanes

Functional gene biomarker

16S rRNA gene biomarker

DhcDhgmdcpA

Anaerobic 1,2-D and 1,2,3-TCP Pathways

1,2-DCP 1,2,3-TCP(Yan et al. 2008; Dhgm strains BLDC-8 and BLDC-9)

(Löffler et al. 1997; Ritalaht et al. 2004; Padilla-Crespo et al. 2013; Dhc

strains RC and KS)

dichloropropane dehalogenase

allyl chloride(unstable)

diallyl sulfide

propene

diallyl disulfideS-

allyl alcohol

Fe2+

S-

S-

Dehalogenimonas lycantrhoporepellens strain BLDC-9

Dichloroelimination to Propene

2H+ + 2e-

2H+ + 2Cl-

Dehalococcoides mccartyi strains KS, RC

SYBR Gold, 1000x

Dichloroelimination of Chloro-Ethanes

Other dichloroelimination reactions involve transformation of TCA to VC and DCA to ethene by Dehalobacter spp.

PCE TCE cis-DCE VC Ethene

1,1,2-TCA

1,2-DCA

Aerobic 1,2,3-TCP Biomarkers and Pathways1,2,3-

trichloropropane

dhaA

hheC

haloalkane dehalogenase

haloalcohol dehalogenase

echAepoxide hydrolase

(Bosma et al. 2002; TCP for Agrobacterium radiobacter strain

AD1)

2,3-dichloro-1-propanol

1,3-dichloro-2-propanol

1-chloro-2,3-epoxypropane 3-chloro-1,2-propanediol

TCP

1,3-DCP

2,3-DCP

3-CPD

1,2-propanediolpropylene glycol

Pseudomonas sp. strain OS-K-29

Pseudomonas sp. strain AD1,Arthrobacter sp. strain AD2, and a coryneform strain AD3

Pseudomonas spp.,

propanediol dehydratase

Salmonella, spp. (needs cobalamin),

biomass, mineralization

pdu

Understanding the CommunityNeed to know accessory organisms that contribute to a specific activity

Dhc requirements:

H2

B12

The Corrin-RingVery costly to make these compounds so why do so?

To find out more about the roles of corrinoids and the populations

that stimulate reductive dechlorination, tune into the

seminar by Dr. Jun Yan, University of Tennessee, this

coming fall.

Yan et al. 2012, Appl. Environ. Microbiol. 78:6630-6636

Yan et al. 2013. Phil. Trans. R. Soc. B. In press

Corrinoid pathway

Value of MBT analysis to MNA sites

Difficult to predict a priori what will happen within an MNA site from only looking at the geochemistry

nutrient limited

may have low O2

often very dilute in contaminant

Studying the bacterial communities allows for a more predictive understanding of the site

This Study: qPCR Data from 869 Wells at 56 Sites

qPCR analysis was putkerformed using established methods at Microbial Insights (at MI/GT/UTK) and geochemical data were compiled by MI/GT/.

Only groundwater samples included

All analyzed for Dhc 16S rRNA gene

Most samples evaluated for RDase genes

Total Bacteria quantified in 337 samples

Ethene data collected at 625 wells

BioTraps deployed in 78 wells at 6 sites

Distribution of Dhc in Groundwater Samples

ND = not detected

Dehalococcoides/L

Treatment Samples (wells) Sites   ND or <103 103 to 104 104 to 106 > 106

TOTAL WELLS 869 56   198 191 275 205Bioaugmentation 92 6 16 15 8 53

Biostimulation 154 20 28 19 43 64MNA 578 54 144 144 202 88

Chem Ox 8 2 0 2 6 0ZVI 37 5 10 11 16 0

Dhc abundance (log gene copies/L)

Ethe

ne c

once

ntra

tion

(ppb

)Lo

g et

hene

con

cent

ratio

n (p

pb)

Dhc abundance (log gene copies/L)

Ethene Formation Correlates with Dhc Abundance

Corresponds with bivariate comparison of ethene to Dhc in GW

n = 176F = 74.8501P = <0.0001R2 = 0.3008significant

Corresponds with bivariate comparison of ethene to Dhc on BioSep Beads (BioTraps)

n = 61F = 22.9258P = <0.0001R2 = 0.279836significant

bvcA+vcrA Gene to Dhc Ratio

Highest ethene production when bvcA+vcrA to Dhc ratio is between 0.05 and 10 in GW

0.05 -10

ZVI

Beads

0.05 -10

Suggests additional VC RDase genes

Despite high ratio of VC-RDases, ethene not observed.

Ethe

ne c

once

ntra

tion

(ppb

)

VC and the bvcA+vcrA Gene to Dhc Ratio

BeadsBeads

0.05 -10 High VC presence correlates with bvcA+ vcrA gene ratios near unity

Wells without bvcA or vcrA but with high numbers of tceA and Dhc

VC c

once

ntra

tion

(ppb

)

RDase Genes can Outnumber Dhc Cells

The most ethene is produced when bvcA+vcrA to Dhc ratio is between 0.05 and 10

When RDase genes >10x Dhc, Dhc abundances tend to be lowUnknown reservoir for additional RDase genes

Dhc to Bacterial 16S rRNA Gene Ratio

Dhc 16S rRNA and VC RDase ratios of 0.0005 - 0.001 (0.05 - 0.1%) of the total Bacterial 16S rRNA gene numbers correlate with ethene detection

ZVI

Dhc 16S rRNA / Bacteria

Ethe

ne (

ppb)

bvcA+vcrA / Bacteria

Ethe

ne (

ppb)

Statistical Correlations with tDCE, cDCE, VC and Ethene

Pairwise Comparison Sample n F value P value R2 significancetDCE to bvcA GW 29 3.5535 0.070 0.116306 weak

cDCE to Dhc GW 338 37.6523 <.0001 0.100768 yescDCE to tceA GW 198 10.9785 <0.0011 0.053042 yescDCE to bvcA GW 109 0.0989 0.754 0.000924 nocDCE tp vcrA GW 136 3.394 0.068 0.024703 weak

VC to Dhc GW 238 75.3012 <0.0001 0.241892 yesBead 35 17.2963 <0.00001 0.343888 yes

VC to tceA GW 173 64.5744 <0.0001 0.274115 yesBead 28 13.6407 0.001 0.344109 yes

VC to bvcA GW 84 0.1949 0.660 0.002372 noBead 27 6.9106 0.014 0.216561 yes

VC to vcrA GW 112 11.6951 <0.0009 0.96101 yesBead 24 6.7682 0.016 0.235268 yes

ethene to Dhc GW 176 74.8501 <0.0001 0.300784 yesBead 61 22.9258 <0.00001 0.279836 yes

ethene to tceA GW 108 73.2656 <0.0001 0.408698 yesBead 41 5.8581 0.020 0.130592 yes

ethene to bvcA GW 77 9.8926 0.002 0.116531 yesBead 42 9.7342 0.003 0.195725 yes

ethene to vcrA GW 110 71.9905 <0.0001 0.399968 yes Bead 37 2.0281 0.163 0.054772 no

Bioaugmentation Consortia

ConclusionsEthene formation correlated with Dhc abundance

Highest ethene formation at sampling locations with near- equal VC RDase to Dhc ratios

High VC presence correlates with bvcA+vcrA gene ratios near unity

RDase genes outnumber Dhc in ≈10% of wells, suggesting an unrecognized reservoir for VC RDase genes

Ethene formation generally occurred when Dhc and VC RDase genes exceeded 0.01% of the total Bacterial 16S rRNA gene abundance

Bioaugmentation inocula infulenced the dominant VC RDase

High Throughput qPCR for Site Analysis

Current approaches are toward high throughput tools with increased sample numbers and replication while monitoring more targets at a reduced cost.

Objective: Design QuantStudio Array

Simultaneous monitoring of 56+ genes for 12-48 samples

Assays target specific microbial processes:Reductive dechlorination (RD-qCHIP)Petroleum remediationMetal reduction Nitrogen cycling… and many more

16S rRNA genes of relevant spp.

Functional genes related to desired activity

QuantStudio 12K Flex System

Microfluidics Robot for loading the chip Thermocycler

Inside the robot

Tips

Tip Waste

Chip holder

384-well sample plate

Analysis

39

33 nL volume

HydrophilicHydrophobic

OpenArray® (Empty) Plate Description

Hydrophilic and hydrophobic coatings enable reagents to stay in the bottomless through- holes via capillary action.

48 subarrays

x 64 through-holes

3072 through-holes per arrayFour plates can be cycled simultaneously, producing >12,000 digital data points per run.

A B C D E F G H I J K L

1 2

3 4

Luc

Bac

16S rRNAgenes

Arch

RDase of known function

Aerobic VC oxidation

Hydrogenase genes

Corrinoid salvageLifeTech array controls

Design Probes and Primers, Verify with qPCR, Decide on Assay Format, and Order Chip.

Example layout for RD-qCHIP

Open Array Workflow

TaqMan assaysordered on-line Primers and probe spotted on

Open Array plateOpen Array plate

Load sampleswith robot

Place lid onarray case Cycle and image up to

4 Open Array plates

Run Plates (UT/ORNL)

Synthesize and load primers and probes to through-wells of the chip: (Life Technologies, Woburn, MA)

Synthesize Array Chip: Overview

Tandem Standard: synthetic DNA of target regions for each assay in KanR vector cloned in E. coli

DhcDhgm

Vector

LucBac

tceAvcrAbvcA

dcpA

Apply in dilution series to verify reproducibility and quantification results of the array

A B C D E F G H I J K L

1 2

3 4

106 copies 104 copies 102 copies

S1 S2

Each spot on the array yields all qPCR data at each cycle and allows replication and dilution of samples with multiple spots

NTCS1(1:10) S2(1:10) Plasmid

Example: Validate RD-qChip with Tandem Standard

Valuable Information from MBTs

Presence/Absence of genes: potential to deal with the compound of interest

Quantitative information of relevance to bioremediation/MNA applications can be obtained with MBTs

Abundance of organisms, genes and transcripts; before, during and after treatment, can be correlated with geochemical data to inform on decision making as to appropriate site remediation action

Final Remarks

If you are interested in great summary of bioremediation applications see Dr. Frank Loeffler’s seminar (Feb, 11, 2013) posted on the MI website.

In Fall 2013 stay tuned to Dr. Jun Yan’s seminar that will go into detail on the contribution of corrinoids and bacterial populations that contribute to organohalide respiration

For case studies on chlorinated ethenes and petroleum bioremediation projects, see the Microbial Insights Website, or contact them for further information at www.microbe.com

Shandra Justicia LeonDarlene WagnerJanet Hatt

Elizabeth PadillaBurcu Simsir

Dora OglesBrett BaldwinAnita Biernacki

Kirsti RitalahtiJun YanCindy Swift

Elizabeth EdwardsAriel GrosternMelanie DuhamelWinnie Chan

To all the people involved in the sampling in the field and in the laboratory analysis of these many samples

To the many collaborators who provided available site characterization information,

To SERDP and ESTCP for providing funding to do this research.

Thank You! Questions